Proteomics in Neuropathic Pain Research
Niederberger, Ellen Ph.D.*; Geisslinger, Gerd M.D., Ph.D.†
Section Editor(s): Warner, David S. M.D.; Warner, Mark A. M.D., Editors
Neuropathic pain is often caused by nerve injury or dysfunction in the peripheral and central nervous system and is frequently associated with allodynia and hyperalgesia. The underlying molecular mechanisms of neuropathic pain are largely unknown, and therefore, pharmacologic treatment is insufficient in many cases. To elucidate translational and posttranslational modifications in the nervous system that arise after nerve injury, a number of proteomic studies have been performed using different animal neuropathy models. The results of these proteomic approaches are summarized in this review to provide a better overview of proteins that are involved into the pathogenesis of nerve injury and neuropathic pain. This might allow a better understanding of the pathophysiologic signaling pathways in this impairment, facilitate the discovery of specific biomarkers, and thus promote the development of novel pain therapies.
is defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system. It can arise from a wide variety of injuries to peripheral or central nerves, including metabolic disorders, traumatic injury, inflammation, and neurotoxicity, and is characterized by spontaneous pain, hyperalgesia (increased pain response to a noxious stimulus), and allodynia (pain elicited by a nonnoxious stimulus), which can persist long after the initial injury is resolved.1
Common causes of neuropathy are diabetes, herpes zoster infections, chronic or acute trauma, and neurotoxins. Furthermore, neuropathic pain occurs frequently in cancer as a direct result of peripheral nerve damage (e.g.
, compression by a tumor) or as a side effect of many chemotherapeutic drugs.
The underlying molecular mechanisms are still not completely understood, and as a consequence, treatment is unsatisfactory in many cases.2–4
Despite the large number of approved analgesics such as opioids or nonsteroidal antiinflammatory drugs, treatment of chronic pain is still often aggravated by poor activity of available drugs and the occurrence of adverse drug reactions.5,6
The current pharmacologic treatment of neuropathic pain includes tricyclic antidepressants such as amitriptyline, anticonvulsants such as gabapentin and pregabalin, serotonin–norepinephrine reuptake inhibitors such as duloxetine, and opioids. However, all of these drugs have limited efficacy combined with a number of side effects, and the mechanism of how they relieve pain is not completely understood. Therefore, there is an urgent need to develop novel therapeutics for an effective treatment of neuropathic pain.
The discovery, design, and evaluation of new drugs are critically dependent on the elucidation of protein mechanisms involved in the respective diseases. Neuropathic pain reflects both peripheral and central sensitization mechanisms which involve transcriptional and posttranscriptional modifications in sensory nerves.1,7
Therefore, proteome analysis in animal models of neuropathy can help to identify pain-related proteins (biomarkers) which may serve as diagnostic markers or drug targets and therefore ameliorate the treatment conditions for patients with neuropathic pain. This review is intended to summarize recent proteomic approaches in animal models of neuropathic pain, which provide several hypotheses about proteins involved into the pathogenesis of this impairment.
Most physiologic body functions are based on the integrity of proteins. Pharmacologically active drugs often target proteins because of their pathophysiologic relevance. However, among more than 3,000 proteins that are suggested as “drugable,” only approximately 500 are indeed targets for pharmacologic therapy so far.8
Proteomics is a term that describes the science and methodology of the investigation of the proteome, which quantitatively acquires the composition of proteins in a cell, a tissue, or an organism. Proteomics is complementary to genomic approaches, which investigate DNA and RNA. Unlike genomics, proteomics delivers information about protein isoforms, posttranslational protein modifications such as glycosylations and phosphorylations, protein–protein interactions, and protein stability and degradation. Whereas the genome of an organism is rather constant, the proteome differs strongly from cell to cell and is constantly modulated through biochemical interactions with the genome and the environment. The protein expression is variable in different parts of a body and depends on a number of parameters, such as age, different environmental conditions, or diseases.
Proteomics can be used for the generation of protein maps of certain tissues (profiling or expression proteomics), for studying pathophysiology by investigation of aberrant proteins (functional proteomics), and for correlating nucleic acid levels with proteins (reviewed by Choudhary and Grant9
or Gorg et al.10
The main technology in proteomics is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), which couples isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension and allows the separation and visualization of complex protein mixtures according to their isoelectric point, molecular weight, solubility, and relative abundance.11
Depending on the pH gradient and the gel size, 2D-PAGE makes it possible to separate thousands of proteins. Protein spots in a gel can be visualized using a variety of chemical stains or fluorescent markers. The intensity of the protein stain is used to quantify protein levels. Subsequent protein analysis and identification are generally performed by mass spectrometry (matrix-assisted laser desorption ionization time-of-flight or surface-enhanced laser desorption ionization time-of-flight mass spectrometry).12
The first dimension of 2D-PAGE is an isoelectric focusing. Proteins are loaded onto gel strips with an immobilized pH gradient and then separated by high-voltage isoelectric focusing according to their net charge (isoelectric point).13
After isoelectric focusing, the immobilized pH gradient strips are placed onto the top of a sodium dodecyl sulfate polyacrylamide gel, and a separation of the proteins according to their molecular weight is performed. Gels are stained with Coomassie brilliant blue, silver, or fluorescence dyes and analyzed for differences in protein expression between two different samples using appropriate image analysis. 2D-PAGE typically allows for the separation of hundreds to thousands of protein spots onto one gel (fig. 1
Identification of 2D-separated Proteins
Protein spots of interest are cut out of the gels and digested to peptides by trypsin. The fragmented peptides are prepared for analysis by extraction with appropriate organic solvents. The proteins are identified by matrix-assisted laser desorption ionization time-of-flight or surface-enhanced laser desorption ionization time-of-flight mass spectrometry on the basis of peptide mass matching.14,15
Clinical Applications of Proteomics
The study of clinical proteomics is suggested as a promising field with potential for many applications, such as identification of biomarkers and monitoring of diseases. Expression proteomics
can be applied for the discovery and validation of diagnostic, prognostic, and predictive biomarkers of diseases, whereas functional proteomics
is intended to identify new targets as well as clarify known drugs. The potential of proteomics has been discovered in various medical areas, in particular cardiovascular diseases, neurologic disorders, infectious diseases, and especially cancer.16
A number of biomarker proteins in several different cancer types have been identified by surface-enhanced laser desorption ionization time-of-flight mass spectrometry and have been reviewed by Engwegen et al.17
By screening already known drugs with proteomic techniques, it is possible to identify proteins that interact with these drugs. These approaches can provide hints about novel targets of the drugs, e.g.
, a proteomic analysis of the protein kinase C inhibitor GF109203X revealed other potential targets, such as cyclin-dependent kinase 2.18
Therefore, in several fields, proteomics will have clinical utility in the future, but to date, the development of proteomics as a technique for high-scale analysis is still in progress. Most approaches are related to cancer diagnosis and treatment. In the context of new drug development, it has to be taken into account that proteomics is mostly the starting point for further investigations which then focus on the most interesting results gained from proteomic studies.
Animal Models of Neuropathic Pain
During the past 10–20 yr, a range of animal models has been established that reflect a variety of different causes for neuropathic pain. They simulate specific human painful conditions, mostly by producing traumatic injuries to the spinal cord or peripheral nerves that result in chronic pain.19
Central Pain Models
Most of the pain models in the central nervous system are based on spinal cord injury (SCI). In patients, this kind of neuropathic pain can be initialized after traumatic or ischemic injury of the spinal cord. Dysesthesia as well as spontaneous and evoked pain are frequently coming along with this neuropathy.
In animals, SCI can be performed by weight drop,20,21
spinal cord compression,22
photochemically induced injury,24,25
excitatory neurotoxin methods,26,27
and spinal hemisection28–30
as well as complete transection.31
These models lead to spontaneous and evoked pain as well as allodynia and hyperalgesia.
Weight Drop Model.
In this quite old model, SCI is produced by dropping a weight on the exposed spinal cord surface at the thoracic–lumbar level, which simulates the human clinical condition of traumatic injury of the spinal cord. This treatment leads to paraplegia and complete segmental necrosis.20,21
Injection of several neurotoxins into the spinal cord can lead to the development of abnormal pain, which is a correlate to pain induced by SCI in humans, including long-lasting spontaneous pain and mechanical and thermal hyperalgesia.26,27
Photochemically Induced Injury.
In this model, spinal cord vessels are damaged by intravenous injection of a photosensitizing dye, which is excitated by an argon ion laser, subsequently leading to parenchymal tissue damage.24
In animals, this treatment leads to autotomy (self-mutilation of the injured paw) and mechanical and cold allodynia as well as hyperalgesia.25
Spinal Cord Hemisection and Complete Transection.
In case of spinal cord hemisection, the spinal cord is only partially dissected at the T13 level, thus leading to an immediate flaccid paralysis of the ipsilateral hind paw that is recovered 15 days after injury. Concurrently, noxious stimulation of this paw induces signs of hyperalgesia and allodynia.28–30
Complete transection of the spinal cord at thoracic level T9–T10 leads to complete paralysis of the hind legs that cannot be regenerated. This model is suitable to investigate the mechanism of the adult inability to regenerate neurons in the central nervous system.31
Neuropathic pain in the periphery can be induced in humans by a variety of events, such as trauma, compression, infections, metabolic diseases, neurotoxins, tumors, and others. Common animal models of peripheral neuropathic pain that simulate human neuropathic conditions include spinal nerve ligation (SNL), partial nerve injury, chronic constriction injury (CCI), and spared nerve injury. Key considerations for the use of these animal models should be that most patients with neuropathic pain resulting from nerve trauma have partial lesion of a nerve and that complete nerve lesions are less frequent.
Spinal Nerve Ligation.
Kim and Chung32,33
reported an experimental model of mononeuropathy with L5 and L6 unilateral (left or right side, respectively) SNL leading to a quick development of hyperalgesia and allodynia (within 24 h) lasting for at least 4 months without autotomy. Deafferentation is moderate in this model. A variant of this model is the ligation of the L5 spinal nerve only,32
which is easier to perform in comparison with combined L5–L6 ligation and also evokes long-lasting allodynia and hyperalgesia. These models simulate the clinical conditions of injury to the nerve plexus or the dorsal root.
Partial Sciatic Nerve Ligation.
To achieve partial nerve injury, 33–50% of the sciatic nerve high in the thigh is unilaterally ligated with silk sutures. Within a few hours after the operation and for several months thereafter, rats develop spontaneous pain characterized by guarding behavior of the ipsilateral hind paw and licking. Furthermore, animals exhibit signs of allodynia to mechanical stimulation and of hyperalgesia to thermal and mechanical noxious stimuli. Autotomy is absent in most cases, and deafferentation is moderate. Behavioral changes and sensory disorders in the partial sciatic nerve ligation model correlate with symptoms of complex regional pain syndrome in humans after peripheral nerve injury.7,34,35
Chronic Constriction Injury of the Sciatic Nerve.
The CCI model has been reported as painful peripheral mononeuropathy where the sciatic nerve is constricted on the left or right side, respectively, through loose ligatures at the mid-thigh level with chromic gut sutures.36,37
This treatment results in nerve inflammation and subsequently leads to a substantial loss of both myelated and unmyelated fibers distal to the placement of the ligatures. Therefore, deafferentation is extensive in this model. Compared with human correlates, the CCI model simulates clinical conditions of chronic nerve compression, which can occur after lumbar disk herniation or nerve entrapment, heavy metal poisoning, anoxia, and metabolic disorders.38,39
Chronic constriction injury rats show behavioral signs of spontaneous pain as well as hyperalgesia due to noxious thermal and mechanical stimuli within the first 24 h after surgery. Furthermore, they develop cold and tactile allodynia. All pain signs last over a period of at least 2 months. Because antiinflammatory treatment of CCI rats results in decreased hyperalgesia,40
it is suggested that this model also comprises an inflammatory component in the development of neuropathic pain.
Experimental Nerve Crush.
In this model, the sciatic nerve is exposed at the mid-thigh level and crushed by hemostatic forceps with grooved jaws. This treatment produces tactile and thermal hyperalgesia and allodynia that manifest first after 3 weeks and last for at least 52 weeks. The nerve crush (NC) leads to wallerian degeneration with subsequent regeneration processes.41,42
Spared Nerve Injury.
The spared nerve injury model of Decosterd and Woolf43
is based on section and ligation of two of the three peripheral branches of the sciatic nerve: The tibial and common peroneal nerves are ligated, and the sural nerve remains intact. It differs from the Chung spinal segmental nerve and the Bennett CCI in that the comingling of distal intact axons with degenerating axons is restricted, and it permits behavioral testing of the noninjured skin territories adjacent to the denervated areas. The spared nerve injury model results in behavioral modifications (sensory hypersensitivity) after less than 24 h that last for at least 6 months. The mechanical and thermal hyperalgesia is increased in the ipsilateral sural and, to a lesser extent, saphenous territories, without any change in heat thermal thresholds.43
Protein Regulations Associated with Neuropathic Pain
The regulation of the protein expression pattern in several tissues of the nervous system has been investigated in a number of aforementioned animal models of neuropathy. Protein modifications differed strongly among the respective models and different tissues, which is in accord with reports showing that most utilized animal neuropathy models show apparent behavioral and morphologic differences.44,45
Furthermore, neuropathic pain in humans also displays different pain syndromes and a number of different causes, thus indicating that a specific nerve injury might have a specific underlying mechanism. An overview of the proteomic studies in different models of nerve injury is given in table 1
Spinal Cord Injury
Differential regulation of proteins in the injured spinal cord of rats has been studied after traumatic injury.46
Among 947 protein spots on the 2D-gel, the authors found more than 39 up-regulated and 29 down-regulated proteins (≥2-fold regulation) 24 h after SCI. Protein alterations at this time point may be related to the onset and early development of neuropathic pain. A number of these protein regulations included neural lineage proteins as well as apoptotic signaling proteins after damage of the spinal cord, which could be confirmed by additional immunohistochemical analysis. Based on the results, it has been concluded that secondary events after SCI include apoptotic cell death as well as regeneration processes by restoring chronic function through increased local levels of growth factors that stimulate the migration, proliferation, gliogenesis, and neurogenesis of endogenous neural progenitor cells in the spinal cord.
Another group performed a model of complete spinal cord transection. This study was mainly focused on investigation of proteins associated with the inability of the adult central nervous system to regenerate. Although axonal regeneration is not suggested to restore the original neuronal anatomy before SCI, it is important for functional recovery by enhancing rewiring of the neuronal network47,48
and will therefore improve symptoms of neuropathy. Five days after injury, an up-regulation of more than 30 proteins (≥1.5-fold regulation) in the spinal cord has been observed. It has been suggested that these proteins are playing various roles in injury and regeneration processes. In particular, two up-regulated proteins (11-zinc-finger protein and glypican) were estimated as inhibitors for axonal growth and regeneration.31
Glypican is a proteoglycan that is expressed in developing immature neurons and prevents axon regeneration as a receptor of axonal growth–inhibiting proteins.49
11-Zinc-finger protein is involved in cell cycle regulation and inhibits cell growth and proliferation in cancer cells.50
Spinal Nerve Ligation
In a model of L5–L6 nerve ligation, five proteins with different expression levels (no minimum regulation threshold indicated) in the spinal cord after nerve injury were identified.51
Among these proteins, the authors focused on creatine kinase B, which was decreased after nerve injury. Because creatine has been found to reduce glutamate levels and to exhibit neuroprotective properties,52–54
the authors concluded that the down-regulation of creatine kinase B might be particularly important for the development and maintenance of neuropathic pain and therefore a valuable therapeutic target for neuropathic pain.
After applying the same neuropathy model, another group investigated the differential protein expression in the brainstem.55
They found 14 up-regulated and 7 down-regulated proteins (≥30% regulation) 7 days after nerve ligation in comparison with sham-operated rats. Interestingly, none of the proteins that have been found regulated in the spinal cord were also regulated in the brainstem and vice versa
, indicating that the nociceptive transmission involves different proteins in the different tissues.
A third study analyzed protein changes in the L4 and L5 dorsal root ganglia after L5 SNL and found 67 regulations (no minimum regulation threshold indicated; proteins are considered as regulated if levels of certain proteins are significantly different from the standard levels [P
< 0.05]) among approximately 1,300 separated proteins.56
Consistent with Lee et al.
a down-regulation of creatine kinase B has been observed; however, this was the only conformity between the different SNL studies.
Sciatic Nerve Crush
The protein expression profile of the rat sciatic nerve has been investigated in a model of experimental NC. The analysis involved immediate responses to injury as well as regeneration processes because tissue was dissected at 5, 10, and 35 days after injury. Among approximately 1,500 spots on each gel, at least 121 regulated proteins (no minimum regulation threshold indicated; proteins are considered as regulated if levels of certain proteins are significantly different from the standard levels [P
< 0.05]) have been found with respect to different time points investigated. A number of these proteins have not been implicated in nerve regeneration previously. Taken together, it was concluded that the detected proteins might reflect the complexity and the temporal aspects of nerve regeneration and, in particular, pronounce the value of glial and inflammatory determinants.41
Partial Nerve Injury
Katano et al.58
investigated the polarity of primary afferent fibers in rats with and without partial nerve injury. They reported the unique expression of 12 proteins in the spinal nerves peripheral to the dorsal root ganglia and 3 in the central region. The proteins in the central region included tubulin β3 and β15, the peripheral proteins collagen α1, α-tubulin, and an isoform of collapsin response mediator 2 (periCRMP-2). The succeeding study was concentrated on characterization of CRMP-2, which was already well known as a regulator of neuronal polarity, axonal growth, and regeneration after nerve injury.34,57
Because total CRMP-2 levels remained unchanged after nerve injury and only peripheral CRMP-2 levels decreased, it has been suggested that periCRMP-2 might be related to pathophysiologic changes in the spinal nerves and regeneration processes in the periphery.58
Chronic Constriction Injury
analyzed the protein expression pattern in the lumbar spinal cord of rats after applying the CCI model. Among an average of 500 protein spots on the 2D-gels, we found 5 significantly regulated protein spots (≥40% regulation) 14 days after induction of CCI. The regulations of proteins in the spinal cord after CCI have been compared with regulations after inflammatory stimulation (zymosan-induced paw inflammation). Only one overlapping regulation could be observed, indicating that inflammatory and neuropathic pain do have distinct regulatory mechanisms.59
Functional Classes of the Identified Proteins
Taken together, all these proteomic studies of neuropathic pain delivered a huge number of proteins that might be involved in the pathogenesis of neuropathy. Although the regulation of the bulk of proteins seems to be unique for the respective neuropathy model and tissue, a number of overlapping protein regulations can be observed by direct comparison of the aforementioned neuropathy studies (table 2
). Interestingly, some proteins are regulated in more than one model—however, not in the same direction. Based on their physiologic function, the regulated proteins can be roughly subdivided into fundamental categories, such as proteins related to cellular homeostasis and metabolism; neuronal function proteins; heat shock proteins, chaperones, and antioxidants; proteins related to cell cycle, apoptosis, and neurodegeneration; signaling proteins; proteins related to the immune system; and proteins related to protein synthesis and processing. These proteins are mostly involved in neuronal degeneration, regeneration, and inflammatory processes. Some of the proteins have already been related to pain, but a number of regulated proteins have not been previously implicated in this context and may therefore provide interesting new fields of pain research.
Proteins Related to Cellular Homeostasis and Metabolism
A great number of proteins involved in cellular metabolism and homeostasis has been regulated after nerve injury. Because these proteins are expressed in almost every cell and play essential roles for the cell functions, it is not likely that these proteins themselves can be used as drug targets. However, it might be of interest that changes in albumin protein levels have been found in three neuropathy models (SCI, NC, and L5 nerve ligation). Altered albumin expressions in tissues of the central nervous system indicate a dysfunction of the blood–brain or blood–spinal cord barrier, respectively. It has been shown that the integrity of this barrier is disturbed after nerve injury resulting in an increased immunoreactivity of albumin in spinal cord tissue.60
Therefore, albumin could be used as a biomarker for certain disturbances of the nervous system.
Neuronal Function Proteins
Most neuronal function proteins are cytoskeleton proteins. The neuronal cytoskeleton is involved in axonal outgrowth and conductivity. Moreover, it plays a pivotal role in signaling from the axon terminals to the cell bodies. This function modulates the intrinsic neuronal capacity for regeneration and repair after nerve injury. The intermediate filament vimentin was up-regulated in three models of neuropathic pain, indicating its importance in neuropathy. It is described that vimentin is generated in the injured nerve axoplasm by local translation and calpain-mediated cleavage and thus allows for the retrograde transport of the phosphorylated mitogen-activated protein kinase Erk. The vimentin–Erk complex is suggested to protect Erk from dephosphorylation, and because the interaction is calcium dependent, the signal generated may provide information about both the injury and the degree of damage as reflected by sustained calcium elevation.61
Heat Shock Proteins, Chaperones, and Antioxidants
The small heat shock protein HSP 27 was regulated after complete spinal cord transection, SCI, NC, and SNL. The heat shock proteins are stress proteins that mediate protein stabilization in various tissues and protect cells from environmental stress. A number of heat shock proteins are up-regulated in the nervous system in response to stress or injury.62,63
Novel evidence suggests that overexpression of the small heat shock protein 27 (Hsp27) in neurons protects against neurotoxic stimuli and may act as an inhibitor of neurodegeneration.64–66
Surprisingly, two of four proteomic studies in neuropathy revealed a down-regulation of Hsp27 after NC and SCI, respectively. This might suggest that the nerves are irreversibly damaged in these models.
Up-regulation of apolipoproteins in response to nerve injury has already been described.67,68
It is suggested that these proteins serve as vehicles to transport lipids between cells during regeneration and degeneration of neurons. A number of apolipoprotein isoforms are regulated in different neuropathic pain models. The isoform A-I was up-regulated in four of the models, indicating a potential role as a biomarker for neuropathic pain.
Proteins Related to Cell Cycle, Apoptosis, and Neurodegeneration
A number of proteins are involved in apoptotic responses which occur frequently after nerve injury in neurons of the peripheral and central nervous system. Apoptosis seems to induce neuronal sensitization and loss of inhibitory systems, and these irreversible processes might be related to the development of neuropathic pain. Prevention of apoptosis might thus suggest future strategies against neuropathic pain.38
Protein disulfide isomerase, a protein that has been related to neuronal apoptosis, was increased after CCI, NC, and SCI. In the nervous system, up-regulation of protein disulfide isomerase has been described as a result of hypoxia and brain ischemia, thus protecting cells from apoptosis.69
The protein acts either as a redox catalyst or as a molecular chaperone that prevents protein aggregation and degradation.70
Neurodegeneration is often accompanied by the formation of toxic protein aggregates, which subsequently induce apoptosis and neuronal loss.71
Therefore, up-regulation of protein disulfide isomerase might constitute a protective mechanism against apoptotic cell death induction in neuropathic pain.
An up-regulation of voltage-dependent anion channels has been found by proteomic analysis in three neuropathy models.31,41,46
These channels are major constituents of the outer mitochondrial membrane, where they control membrane permeability and the subsequent release of apoptosis promoting factors.72
Therefore, in the context of nerve injury, they might be involved in the degradation of neurons.
Proteins Related to the Immune System
Galectin 3 is a galactose-specific lectin that was regulated in three proteomic studies. The protein is involved in neuronal cell adhesion and neurite outgrowth.73
After peripheral nerve injury in rats, an N
-methyl-d-aspartate–mediated up-regulation of galactin 3 has been observed in subpopulations of dorsal horn neurons.74
Because galectin is also involved in myelin phagocytosis and therefore in wallerian degeneration of neurons, it might serve as a trigger for neuronal apoptosis induction after nerve injury.75
Current Limitations of Proteomic Research
At the moment, a number of limitations occurring with proteomics in neuroscience still hinder the analysis of the complete protein spectrum. At first, sample preparation of brain or spinal cord delivers a complex and heterogenous mix of cells, which cannot be distinguished on 2D-gels. Single, defined cells can only be investigated from neuronal cell cultures. Laser microdissection of single cells might yield a new method to analyze small groups of cells from neuronal tissues. Second, low-level regulatory proteins cannot be detected; protein analysis is substrate limited because no amplification methods are available as yet. Similarly, high- and low-molecular-weight proteins as well as hydrophobic membrane proteins are difficult to separate by 2D-gel electrophoresis. Hence, the great advantage of 2D-PAGE is the evaluation of limited protein patterns under physiologic and pathophysiologic conditions, respectively. This functional proteomic analysis can already deliver a substantial volume of data, which might serve as a basis for the development of further research approaches.9,76
The treatment of pathologic pain coming along with neuropathy requires efficient and highly specific drugs. However, patients are often not adequately treated by the currently available drugs. Therefore, it is necessary to gain more insights into the molecular mechanisms of neuropathy and to find proteins as drug targets that are specifically regulated in neuropathic pain.77
A number of animal models of neuropathy have been developed for research of neuropathic pain which result in a highly reproducible and frequent development of allodynia and hyperalgesia. These models differ strongly among each other by reflecting central or peripheral nerve injury as well as different approaches to produce nerve damage. Therefore, it is difficult to judge which model is the best reflection of the “normal” human response to nerve injury, and data of the different models must be interpreted in the context of the specific pain model.
Despite the importance of collecting new data about signaling pathways in pain, only a few studies have been designed to investigate differences in protein patterns in the nervous system after neuropathic pain. However, these studies revealed a large amount of regulated proteins. Depending on the experimental setting, there were huge differences among the protein regulations, which might be due to different neuropathy models or different neuronal tissues. Furthermore, it should be taken into account that the different approaches to investigate the protein pattern are very heterogeneous. The differential sample preparations, various time points for tissue dissection, differing conditions for isoelectric focusing (pH range, separation protocol, and so on) and sodium dodecyl sulfate polyacrylamide gel electrophoresis might also contribute to the wide variety of regulated proteins.
In principle, proteomics can deliver useful information regarding “pain-associated” proteins and may therefore provide a reasonable technique for the identification of regulated proteins in the nervous system after nerve injury.
Therefore, the studies summarized here, together with previous studies that identified single molecular participants in neuropathic pain, may lead to a better understanding of the molecular mechanisms that are involved in these processes. This might help to control the pathophysiologic signaling pathways in pain and thus promote the development of new pain therapeutics by using subsequent systematic investigations.
1. Woolf CJ, Mannion RJ: Neuropathic pain: Aetiology, symptoms, mechanisms, and management. Lancet 1999; 353:1959–64
2. Sindrup SH, Jensen TS: Efficacy of pharmacological treatments of neuropathic pain: An update and effect related to mechanism of drug action. Pain 1999; 83:389–400
3. Hansson PT, Dickenson AH: Pharmacological treatment of peripheral neuropathic pain conditions based on shared commonalities despite multiple etiologies. Pain 2005; 113:251–4
4. Chen H, Lamer TJ, Rho RH, Marshall KA, Sitzman BT, Ghazi SM, Brewer RP: Contemporary management of neuropathic pain for the primary care physician. Mayo Clin Proc 2004; 79:1533–45
5. Kingery WS: A critical review of controlled clinical trials for peripheral neuropathic pain and complex regional pain syndromes. Pain 1997; 73:123–39
6. Koltzenburg M: Painful neuropathies. Curr Opin Neurol 1998; 11:515–21
7. Campbell JN, Meyer RA: Mechanisms of neuropathic pain. Neuron 2006; 52:77–92
8. Orth AP, Batalov S, Perrone M, Chanda SK: The promise of genomics to identify novel therapeutic targets. Expert Opin Ther Targets 2004; 8:587–96
9. Choudhary J, Grant SG: Proteomics in postgenomic neuroscience: The end of the beginning. Nat Neurosci 2004; 7:440–5
10. Gorg A, Weiss W, Dunn MJ: Current two-dimensional electrophoresis technology for proteomics. Proteomics 2004; 4:3665–85
11. O’Farrell PH: High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975; 250:4007–21
12. Matsumoto H, Komori N: Protein identification on two-dimensional gels archived nearly two decades ago by in-gel digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Anal Biochem 1999; 270:176–9
13. Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W: The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000; 21:1037–53
14. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P: In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 1992; 203:173–9
15. Burre J, Beckhaus T, Corvey C, Karas M, Zimmermann H, Volknandt W: Synaptic vesicle proteins under conditions of rest and activation: Analysis by 2-D difference gel electrophoresis. Electrophoresis 2006; 27:3488–96
16. Banks RE, Dunn MJ, Hochstrasser DF, Sanchez JC, Blackstock W, Pappin DJ, Selby PJ: Proteomics: New perspectives, new biomedical opportunities. Lancet 2000; 356:1749–56
17. Engwegen JY, Gast MC, Schellens JH, Beijnen JH: Clinical proteomics: Searching for better tumour markers with SELDI-TOF mass spectrometry. Trends Pharmacol Sci 2006; 27:251–9
18. Brehmer D, Godl K, Zech B, Wissing J, Daub H: Proteome-wide identification of cellular targets affected by bisindolylmaleimide-type protein kinase C inhibitors. Mol Cell Proteomics 2004; 3:490–500
19. Wang LX, Wang ZJ: Animal and cellular models of chronic pain. Adv Drug Deliv Rev 2003; 55:949–65
20. Greenberg J, McKeever PE, Balentine JD: Lysosomal activity in experimental spinal cord trauma: An ultrastructural cytochemical evaluation. Surg Neurol 1978; 9:361–4
21. Anderson TE: A controlled pneumatic technique for experimental spinal cord contusion. J Neurosci Methods 1982; 6:327–33
22. Tarlov IM: Acute spinal cord compression paralysis. J Neurosurg 1972; 36:10–20
23. Rivlin AS, Tator CH: Effect of duration of acute spinal cord compression in a new acute cord injury model in the rat. Surg Neurol 1978; 10:38–43
24. Watson BD, Prado R, Dietrich WD, Ginsberg MD, Green BA: Photochemically induced spinal cord injury in the rat. Brain Res 1986; 367:296–300
25. Hao JX, Xu XJ, Aldskogius H, Seiger A, Wiesenfeld-Hallin Z: Photochemically induced transient spinal ischemia induces behavioral hypersensitivity to mechanical and cold stimuli, but not to noxious-heat stimuli, in the rat. Exp Neurol 1992; 118:187–94
26. Gorman AL, Yu CG, Ruenes GR, Daniels L, Yezierski RP: Conditions affecting the onset, severity, and progression of a spontaneous pain-like behavior after excitotoxic spinal cord injury. J Pain 2001; 2:229–40
27. Wilcox GL: Pharmacological studies of grooming and scratching behavior elicited by spinal substance P and excitatory amino acids. Ann N Y Acad Sci 1988; 525:228–36
28. Christensen MD, Hulsebosch CE: Chronic central pain after spinal cord injury. J Neurotrauma 1997; 14:517–37
29. Christensen MD, Everhart AW, Pickelman JT, Hulsebosch CE: Mechanical and thermal allodynia in chronic central pain following spinal cord injury. Pain 1996; 68:97–107
30. Bennett AD, Chastain KM, Hulsebosch CE: Alleviation of mechanical and thermal allodynia by CGRP(8-37) in a rodent model of chronic central pain. Pain 2000; 86:163–75
31. Ding Q, Wu Z, Guo Y, Zhao C, Jia Y, Kong F, Chen B, Wang H, Xiong S, Que H, Jing S, Liu S: Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury. Proteomics 2006; 6:505–18
32. Kim SH, Chung JM: An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992; 50:355–63
33. Choi Y, Yoon YW, Na HS, Kim SH, Chung JM: Behavioral signs of ongoing pain and cold allodynia in a rat model of neuropathic pain. Pain 1994; 59:369–76
34. Seltzer Z, Dubner R, Shir Y: A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 1990; 43:205–18
35. Tahmoush AJ: Causalgia: Redefinition as a clinical pain syndrome. Pain 1981; 10:187–97
36. Bennett GJ, Xie YK: A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988; 33:87–107
37. George A, Marziniak M, Schafers M, Toyka KV, Sommer C: Thalidomide treatment in chronic constrictive neuropathy decreases endoneurial tumor necrosis factor-alpha, increases interleukin-10 and has long-term effects on spinal cord dorsal horn met-enkephalin. Pain 2000; 88:267–75
38. Zimmermann M: Pathobiology of neuropathic pain. Eur J Pharmacol 2001; 429:23–37
39. Mochizucki D: Serotonin and noradrenaline reuptake inhibitors in animal models of pain. Hum Psychopharmacol 2004; 19 (suppl 1):S15–9
40. Wagner R, Janjigian M, Myers RR: Anti-inflammatory interleukin-10 therapy in CCI neuropathy decreases thermal hyperalgesia, macrophage recruitment, and endoneurial TNF-alpha expression. Pain 1998; 74:35–42
41. Jimenez CR, Stam FJ, Li KW, Gouwenberg Y, Hornshaw MP, De Winter F, Verhaagen J, Smit AB: Proteomics of the injured rat sciatic nerve reveals protein expression dynamics during regeneration. Mol Cell Proteomics 2005; 4:120–32
42. Bester H, Beggs S, Woolf CJ: Changes in tactile stimuli-induced behavior and c-Fos expression in the superficial dorsal horn and in parabrachial nuclei after sciatic nerve crush. J Comp Neurol 2000; 428:45–61
43. Decosterd I, Woolf CJ: Spared nerve injury: An animal model of persistent peripheral neuropathic pain. Pain 2000; 87:149–58
44. Kim KJ, Yoon YW, Chung JM: Comparison of three rodent neuropathic pain models. Exp Brain Res 1997; 113:200–6
45. Lee DH, Chung K, Chung JM: Strain differences in adrenergic sensitivity of neuropathic pain behaviors in an experimental rat model. Neuroreport 1997; 8:3453–6
46. Kang SK, So HH, Moon YS, Kim CH: Proteomic analysis of injured spinal cord tissue proteins using 2-DE and MALDI-TOF MS. Proteomics 2006; 6:2797–812
47. Beattie MS, Farooqui AA, Bresnahan JC: Review of current evidence for apoptosis after spinal cord injury. J Neurotrauma 2000; 17:915–25
48. Profyris C, Cheema SS, Zang D, Azari MF, Boyle K, Petratos S: Degenerative and regenerative mechanisms governing spinal cord injury. Neurobiol Dis 2004; 15:415–36
49. Hagino S, Iseki K, Mori T, Zhang Y, Hikake T, Yokoya S, Takeuchi M, Hasimoto H, Kikuchi S, Wanaka A: Slit and glypican-1 mRNAs are coexpressed in the reactive astrocytes of the injured adult brain. Glia 2003; 42:130–8
50. Rasko JE, Klenova EM, Leon J, Filippova GN, Loukinov DI, Vatolin S, Robinson AF, Hu YJ, Ulmer J, Ward MD, Pugacheva EM, Neiman PE, Morse HC III, Collins SJ, Lobanenkov VV: Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF. Cancer Res 2001; 61:6002–7
51. Lee SC, Yoon TG, Yoo YI, Bang YJ, Kim HY, Jeoung DI, Kim HJ: Analysis of spinal cord proteome in the rats with mechanical allodynia after the spinal nerve injury. Biotechnol Lett 2003; 25:2071–8
52. Sullivan PG, Geiger JD, Mattson MP, Scheff SW: Dietary supplement creatine protects against traumatic brain injury. Ann Neurol 2000; 48:723–9
53. Klivenyi P, Ferrante RJ, Matthews RT, Bogdanov MB, Klein AM, Andreassen OA, Mueller G, Wermer M, Kaddurah-Daouk R, Beal MF: Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis. Nat Med 1999; 5:347–50
54. Xu CJ, Klunk WE, Kanfer JN, Xiong Q, Miller G, Pettegrew JW: Phosphocreatine-dependent glutamate uptake by synaptic vesicles: A comparison with ATP-dependent glutamate uptake. J Biol Chem 1996; 271:13435–40
55. Alzate O, Hussain SR, Goettl VM, Tewari AK, Madiai F, Stephens RL Jr, Hackshaw KV: Proteomic identification of brainstem cytosolic proteins in a neuropathic pain model. Brain Res Mol Brain Res 2004; 128:193–200
56. Komori N, Takemori N, Kim HK, Singh A, Hwang SH, Foreman RD, Chung K, Chung JM, Matsumoto H: Proteomics study of neuropathic and nonneuropathic dorsal root ganglia: Altered protein regulation following segmental spinal nerve ligation injury. Physiol Genomics 2007; 29:215–30
57. Arimura N, Menager C, Fukata Y, Kaibuchi K: Role of CRMP-2 in neuronal polarity. J Neurobiol 2004; 58:34–47
58. Katano T, Mabuchi T, Okuda-Ashitaka E, Inagaki N, Kinumi T, Ito S: Proteomic identification of a novel isoform of collapsin response mediator protein-2 in spinal nerves peripheral to dorsal root ganglia. Proteomics 2006; 6:6085–94
59. Kunz S, Tegeder I, Coste O, Marian C, Pfenninger A, Corvey C, Karas M, Geisslinger G, Niederberger E: Comparative proteomic analysis of the rat spinal cord in inflammatory and neuropathic pain models. Neurosci Lett 2005; 381:289–93
60. Gordh T, Chu H, Sharma HS: Spinal nerve lesion alters blood-spinal cord barrier function and activates astrocytes in the rat. Pain 2006; 124:211–21
61. Perlson E, Hanz S, Ben-Yaakov K, Segal-Ruder Y, Seger R, Fainzilber M: Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve. Neuron 2005; 45:715–26
62. Sharma HS, Gordh T, Wiklund L, Mohanty S, Sjoquist PO: Spinal cord injury induced heat shock protein expression is reduced by an antioxidant compound H-290/51: An experimental study using light and electron microscopy in the rat. J Neural Transm 2006; 113:521–36
63. Yenari MA, Giffard RG, Sapolsky RM, Steinberg GK: The neuroprotective potential of heat shock protein 70 (HSP70). Mol Med Today 1999; 5:525–31
64. Costigan M, Mannion RJ, Kendall G, Lewis SE, Campagna JA, Coggeshall RE, Meridith-Middleton J, Tate S, Woolf CJ: Heat shock protein 27: Developmental regulation and expression after peripheral nerve injury. J Neurosci 1998; 18:5891–900
65. Kretz A, Schmeer C, Tausch S, Isenmann S: Simvastatin promotes heat shock protein 27 expression and Akt activation in the rat retina and protects axotomized retinal ganglion cells in vivo
. Neurobiol Dis 2006; 21:421–30
66. Latchman DS: HSP27 and cell survival in neurones. Int J Hyperthermia 2005; 21:393–402
67. Boyles JK, Notterpek LM, Anderson LJ: Accumulation of apolipoproteins in the regenerating and remyelinating mammalian peripheral nerve: Identification of apolipoprotein D, apolipoprotein A-IV, apolipoprotein E, and apolipoprotein A-I. J Biol Chem 1990; 265:17805–15
68. Ignatius MJ, Gebicke-Harter PJ, Skene JH, Schilling JW, Weisgraber KH, Mahley RW, Shooter EM: Expression of apolipoprotein E during nerve degeneration and regeneration. Proc Natl Acad Sci U S A 1986; 83:1125–9
69. Tanaka S, Uehara T, Nomura Y: Up-regulation of protein-disulfide isomerase in response to hypoxia/brain ischemia and its protective effect against apoptotic cell death. J Biol Chem 2000; 275:10388–93
70. Gruber CW, Cemazar M, Heras B, Martin JL, Craik DJ: Protein disulfide isomerase: The structure of oxidative folding. Trends Biochem Sci 2006; 31:455–64
71. Hinault MP, Ben-Zvi A, Goloubinoff P: Chaperones and proteases: cellular fold-controlling factors of proteins in neurodegenerative diseases and aging. J Mol Neurosci 2006; 30:249–65
72. Granville DJ, Gottlieb RA: The mitochondrial voltage-dependent anion channel (VDAC) as a therapeutic target for initiating cell death. Curr Med Chem 2003; 10:1527–33
73. Pesheva P, Kuklinski S, Schmitz B, Probstmeier R: Galectin-3 promotes neural cell adhesion and neurite growth. J Neurosci Res 1998; 54:639–54
74. Cameron AA, Dougherty PM, Garrison CJ, Willis WD, Carlton SM: The endogenous lectin RL-29 is transynaptically induced in dorsal horn neurons following peripheral neuropathy in the rat. Brain Res 1993; 620:64–71
75. Reichert F, Saada A, Rotshenker S: Peripheral nerve injury induces Schwann cells to express two macrophage phenotypes: Phagocytosis and the galactose-specific lectin MAC-2. J Neurosci 1994; 14:3231–45
76. Lubec G, Krapfenbauer K, Fountoulakis M: Proteomics in brain research: Potentials and limitations. Prog Neurobiol 2003; 69:193–211
77. Scholz J, Woolf CJ: Can we conquer pain? Nat Neurosci 2002; 5 (suppl):1062–7
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